1. Field of the Invention
This invention relates to a device and method for separating blood cells from biological fluids and, subsequently, assaying for a constitutent in the remaining fluid, and particularly separating blood cells from whole blood and assaying for a constitutent that may be present in the plasma. Specifically, this invention relates to a self-contained device and method for analyzing very small samples of whole blood either semi-quantitatively or qualitatively.
2. Description of Related Art Including Information Disclosure Statement
Medicine has growing demands for quick, accurate determination of analytes in biological fluids and especially blood plasma. Traditionally, assays for analytes have been performed by laboratories and required skilled technicians, complex apparatuses, multi-step procedures requiring a plurality of reagents, and considerable time in order to determine results.
Numerous qualitative and some quantitative devices and methods have been developed which eliminate or decrease the need for skilled technicians in the analysis of analytes in blood plasma. Many of these devices and methods are test strips or dip sticks which, when exposed to blood, plasma, or other body fluids, measure an analyte in order to obtain a diagnostic result. A common example of this technology includes the various test products for determining the level of blood glucose in diabetics. These tests are conducted by comparing the amount of color formation of the dip stick or paper strip to a standard or color chart. The convenience of many of these tests does not always eliminate their complexity nor the requirement that the test be performed by skilled, medical personnel.
Analytes in whole blood are, ordinarily, measured after the red blood cells are separated from the plasma. This separation is particularly important with detection methods that depend on a color change. The presence of red blood cells in these tests interferes with the color determination. The red blood cells can also interfere with the measurement of an analyte by participating in a chemical reaction with the test reagents.
Accepted technologies for separating plasma from blood cells include centrifugation, agglutination, and filtration. These technologies, typically, require manual operations and, because they have several steps, can introduce health risks that are associated with the handling of blood and blood products. In the case of centrifugation, the equipment is expensive. Filtration is slow and wastes sample because cells tend to collect in the filter pores and impede the passage of plasma through the filter.
Methods that require a transfer of blood sample from one device to another can require relatively large sample volumes. These sample volumes can be on the order of milliliters and are required in part because of sample losses that occur during the transfer of blood. A sample of this volume requires collection by venipuncture rather than a skin prick. Venipuncture is undesirable because it is more invasive to the patient and requires skill in order to be safely performed.
A number of products have been developed that measure analytes in whole blood whereby plasma separation occurs within the device. Most of these products filter the red blood cells from plasma. Such products include the Syntex product sold under the trade name "Acculevel," the Chematic Inc. products sold under the trade name "Chemcard," the Ames Blood Glucose Sticks product, the Reflatron Cholesterol test product, the Hybritech product sold under the trade name "ICON/CITE," and the Abbott product sold under the trade name "TestPack." Commercial blood analyte test products have the undesirable characteristics of producing inconsistent results or requiring relatively large blood samples. Inconsistent results can be either variations between various test kits in actual readings of an analyte from a common blood sample or variations in the color development of a chromatic reaction within a single test kit.
Inconsistent test results are sometime caused by incomplete separation of blood cells and plasma. The presence of red blood cells in the reagents causes inaccurate measurement of the plasma sample and, in the case of glucose sticks, makes additional procedural manipulations necessary. Many products do not have efficient flow of plasma through the blood separation portion of the device. This results in slow test results and in uneven application or exposure of sample to the reagents and, therefore, uneven reaction color.
The requirement by certain commercial products for relatively large blood samples can be due to many factors. One such factor is that plasma is often retained in the blood cell and plasma separation element of a device. This requires a relatively large blood sample be drawn from the patient in order to provide sufficient plasma to the reagent zone of the device. For example, many of these commercial devices require "several drops" or from 200 to 300 .mu.l of whole blood. A small skin prick by a needle, typically, is not satisfactory to obtain this volume of blood. In contrast, the product sold under the trade name "Acculevel" requires only a small sample of precisely 35 .mu.l. Dispensing such a small sample requires manual dexterity and practice. Therefore, this test does not lend itself to use by lay persons.
U.S. Pat. No. 3,552,925 to Fetter discloses a method for separating whole blood into a substantially colorless fluid and the red cell components or residue. Whole blood is contacted with a matrix containing a water-soluble salt having an inorganic cation such as potassium citrate, ammonium sulfate, zinc sulfate, or the like. A preferred embodiment has a matrix containing one of these salts positioned adjacent to a reaction layer. The inventors do not disclose use of any substance other than inorganic salts to separate the red blood cells.
U.S. Pat. No. 4,594,327 to Zuk discloses an assay method for whole blood samples. This invention uses at least one specific binding pair which is substantially uniformly bound to a solid bibulous element. The method of using this invention requires that the blood sample be mixed with an agent that binds red blood cells, and possibly other ingredients, to form an assay medium. When the analyte is being drawn into the device, blood cells aggregate at an air-liquid interface. This invention also requires a signal producing system in order to evaluate the rest results and does not provide a self-contained unit that can semi-quantitatively determine concentration of an analyte in a single step.
U.S. Pat. No. 4,477,575 to Vogel et al. discloses a process and composition for separating plasma from whole blood. The composition includes glass fibers having an average diameter of from 0.2 to 5 microns and a density of 0.1 to 0.5 grams per cubic centimeter. The process includes the steps of slowly trickling whole blood onto one side of a layer composed of a composition of glass fibers whereby plasma separated from the blood becomes available at another side of the layer. The total volume of the plasma separated from the blood is limited to at most 50% of the void volume of the glass fiber layer. The glass fiber layer is removed after the plasma has had time to enter the reaction layer. Separation can be improved by adding an absorbent layer that wicks plasma to a reaction layer. However, this increases the sample volume and time required to perform a test. The glass fibers of this device rely on filtration. Therefore, serum is trapped in the device and separation efficiency is poor.
U.S. Pat. No. 4,678,757 to Rapkin et al. discloses a device and method for whole blood separation and analysis. The whole blood is introduced to a carrier containing a carbohydrate layer which rapidly separates fluid from cellular fractions. In a preferred embodiment, the device is fabricated in a sandwich design containing layers of carbohydrate and reagent material between two layers of plastic. Various configurations are described and several provide multiple analyses per device. Permeable or impermeable carriers can also be used. The preferred permeable carriers are "absorbent materials," such as filter paper, felts, and fleeces. Carbohydrate solutions are applied and permeable carrier dried. The carbohydrate with impermeable carriers is applied to the carrier as a suspended powder. The preferred embodiment of this invention uses absorbent materials that reduce the amount of plasma that is released for a given volume of a whole blood sample.
U.S. Pat. No. 4,696,797 to Kelton discloses a blood separating device wherein a filter body is used having a porosity small enough to physically trap blood particles on the filter.
U.S. Pat. No. 3,983,005 to Goodhue et al. discloses a device for the analysis of cholesterol. The device has at least two layers. One layer spreads the sample and the other layer contains reagents for the analysis. Cholesterol oxidase and cholesterol ester hydrolyzing components can be present in either of these two layers. The use of this device requires the prior separation of blood cells from a whole blood sample.
U.S. Pat. Nos. 3,964,871 and 4,042,329 to Hochstrasser disclose methods and devices for detecting glucose and cholesterol. The devices of these disclosures are dipped into a sample of essentially cell-free body fluid. The device has a built-in color intensity scale. The distance along the scale for which there is a color change is directly correlated to analyte concentration and allows a semi-quantitative determination of analyte. This device requires a separation of plasma from whole blood before use and requires a blood sample on the order of milliliters rather than microliters in order to perform the test.
U.S. Pat. No. 4,160,008 to Fenochetti describes a device that analyzes a sample for several analytes on a common support. Each test region is isolated above the common support. A blotter is provided on the support to insure against run-off and cross-contaimination of analytical reagents.
U.S. Pat. No. 4,435,504 to Zuk et al. discloses an immunochromatographic assay with a support having bound "MIP" or antibody and a second enzyme. This invention measures the amount of analyte in a sample solution of a body fluid. This measurement is conducted by combining a premeasured volume of sample with a premeasured volume of a solution of enzyme labelled analyte and immunochromatographing the solution or employing a combination of enzymes wherein one enzyme is label and the other enzyme is affixed to the chromatographic support. The assay of this invention is performed by contacting the immunochromatograph with the sample containing solution. The sample traverses a region of the immunochromatograph by elution or solvent transport. The device used in this assay has a region in which the antibody is non-diffusively bound to a bibulous support. The analyte from the same and its enzyme labelled conjugate traverses this zone along with the solvent. The analyte becomes bound to the support through the intermediacy of antibody complex formation. The signal producing system provides the area in this region with a color change which identifies the distance from a predetermined point over which the analyte and its enzyme labelled conjugate have traveled. In this manners a quantitative determination of the analyte can be made. This invention does not directly test whole blood and requires accurate volumetric measurement of the blood sample and the enzyme conjugate solution and dilution of the blood sample by a separately applied solvent. Furthermore, the determination of the analyte concentration with this invention requires a "signal producing system" involving the absorption or emission or electromagnetic radiation such as ultraviolet light. The invention of this disclosure does not provide an immediate determination of the concentration of an analyte.
An article by Sloan et al. discloses "The Quantab Strip in the Measurement of Urinary Chloride and Sodium Concentrations" Clin. Chem. 30 (10), 1705-1707 (1984). The test strip of this disclosure provides a quantitative measurement of chloride and sodium concentrations in urine. The test strips of this invention rely on wicking alone, and typically require 15 to 20 minutes to fill the measurement zone. This device does not provide a rapid test nor a means for separation of cells.
Another area of art related to this invention is the elimination of blotchiness in color development of analyte tests. Blotchiness is a common problem with many devices. A representative example of this problem is sometimes displayed by the product sold under the trade name "Chemicard" which is manufactured by Chematics in North Webster, Indiana. This test conveniently measures cholesterol in three minutes from a drop of whole blood. The developed color readings of this product are sometimes blotchy and, therefore, difficult to read.
The product sold under the trade name "Chemicard" uses a drop of blood in a test area. The test area is a small porous pad supported at its periphery by a card that covers most of the device. A membrane is attached beneath the test area to a small support. The small support is also attached to the top card. The membrane keeps most blood cells from reaching the reaction pad. The reaction pad is supported by a card and surrounded by a color wheel. After three minutes, the top card and membrane are removed and the reaction pad compared with the color wheel. Six color intensities for cholesterol levels ranging from 150 to 300 mg/dl are indicated by this test. A significant number of users of this product in an independent survey expressed dissatisfaction with readability of the results of the test because the developed color is so blotchy.
The industry lacks an accurate self-contained means for analyzing of plasma constitutents that uses a small quantity of whole blood and that is fast and accurate and can be used by the patient for self-testing.